This project will study the cell-mediated rejection of xenogeneic tissues. Preliminary studies have shown that rejection of discordant xenogeneic skin grafts by mice is mediated primarily by CD4+ T cells, that direct murine T cell responses to xenoantigens in vitro are extremely weak, but that nonetheless, mice still reject xenogeneic skin grafts in vivo more vigorously than they do allografts. These findings suggest that our current in vitro assays measure the wrong T cell response, that the mechanisms of T-cell mediated xenograft rejection may differ from those of allografts, and that either different forms of immunosuppression or selective genetic modifications of xenogeneic donors will be needed to inhibit T cell-mediated rejection of xenogeneic heart transplants. The primary issue addresses in these studies is whether the greater strength of cell-mediated xenogeneic immunity resides in the indirect pathway (Specific Aim#1), by which the peptides of foreign antigens are presented by recipient MHC molecules, or in the direct pathway (Specific Aim#2), by which xenogeneic antigens are recognized on the donor's antigen presenting cells. The proposed experiments make use of several new genetically-altered mouse strains which enable us to investigate these mechanisms of graft rejection separately. To extend the results of our mouse studies to more clinically-relevant species combinations, we will perform experiments in Specific Aim#3 to examine the T cell responses of primates to pig antigens. In addition to in vitro assays, we will adoptively transfer primate lymphocytes to RAG-1 knock out mice to study direct versus indirect rejection of xenografts in vivo. Finally, we will study the T cell-mediated resistance to xenogeneic bone marrow engraftment in Specific Aim#4 in collaboration with Project#4. We will use the genetically-altered mouse strains to determine whether direct or indirect T cell responses are primarily responsible for rejection of xenogeneic bone marrow. The fundamental purpose of these experiments is to identify modifications of our tolerance induction protocol that will achieve more successful engraftment of xenogeneic bone marrow and long-term survival of xenogeneic organs in large animals. Thus, even though the proposed studies in this Project begin with some basic studies in cellular immunology in mice, their object is directly related to the goal of this Program Project.